NZ246211A - Osteoblast growth and bone enhancing factor(s) derived from whey and foods containing it - Google Patents
Osteoblast growth and bone enhancing factor(s) derived from whey and foods containing itInfo
- Publication number
- NZ246211A NZ246211A NZ246211A NZ24621192A NZ246211A NZ 246211 A NZ246211 A NZ 246211A NZ 246211 A NZ246211 A NZ 246211A NZ 24621192 A NZ24621192 A NZ 24621192A NZ 246211 A NZ246211 A NZ 246211A
- Authority
- NZ
- New Zealand
- Prior art keywords
- whey
- molecular weight
- bone
- osteoblast growth
- bone enhancing
- Prior art date
Links
- 108010046377 Whey Proteins Proteins 0.000 title claims abstract description 123
- 210000000988 bone and bone Anatomy 0.000 title claims abstract description 98
- 102000007544 Whey Proteins Human genes 0.000 title claims abstract description 82
- 210000000963 osteoblast Anatomy 0.000 title claims abstract description 73
- 239000005862 Whey Substances 0.000 title claims abstract description 52
- 235000013305 food Nutrition 0.000 title claims abstract description 39
- 230000002708 enhancing effect Effects 0.000 title claims description 65
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 114
- 235000021119 whey protein Nutrition 0.000 claims abstract description 65
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 43
- 239000002244 precipitate Substances 0.000 claims abstract description 42
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 38
- 239000007864 aqueous solution Substances 0.000 claims abstract description 23
- 239000011780 sodium chloride Substances 0.000 claims abstract description 19
- 239000006228 supernatant Substances 0.000 claims abstract description 14
- 239000012466 permeate Substances 0.000 claims abstract description 13
- 239000000126 substance Substances 0.000 claims description 21
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims description 16
- 238000010438 heat treatment Methods 0.000 claims description 15
- 238000001914 filtration Methods 0.000 claims description 6
- 230000001376 precipitating effect Effects 0.000 claims description 6
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 238000000034 method Methods 0.000 claims 1
- 239000003814 drug Substances 0.000 abstract description 8
- 238000000108 ultra-filtration Methods 0.000 abstract description 2
- 206010057178 Osteoarthropathies Diseases 0.000 abstract 1
- 230000001737 promoting effect Effects 0.000 abstract 1
- 230000003014 reinforcing effect Effects 0.000 abstract 1
- 238000005194 fractionation Methods 0.000 description 32
- 239000000047 product Substances 0.000 description 29
- 239000000243 solution Substances 0.000 description 29
- 239000000843 powder Substances 0.000 description 24
- 241000700159 Rattus Species 0.000 description 22
- 230000000694 effects Effects 0.000 description 22
- 210000004080 milk Anatomy 0.000 description 19
- 239000008267 milk Substances 0.000 description 19
- 235000013336 milk Nutrition 0.000 description 18
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 16
- 229960005069 calcium Drugs 0.000 description 16
- 239000011575 calcium Substances 0.000 description 16
- 229910052791 calcium Inorganic materials 0.000 description 16
- 208000001132 Osteoporosis Diseases 0.000 description 13
- 108090000765 processed proteins & peptides Proteins 0.000 description 13
- 102000004196 processed proteins & peptides Human genes 0.000 description 13
- 239000012141 concentrate Substances 0.000 description 12
- 235000008504 concentrate Nutrition 0.000 description 12
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 8
- 229940079593 drug Drugs 0.000 description 7
- 238000001223 reverse osmosis Methods 0.000 description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- 239000005018 casein Substances 0.000 description 6
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 6
- 235000021240 caseins Nutrition 0.000 description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 210000000540 fraction c Anatomy 0.000 description 6
- 102000008186 Collagen Human genes 0.000 description 5
- 108010035532 Collagen Proteins 0.000 description 5
- 229910019142 PO4 Inorganic materials 0.000 description 5
- 108091005804 Peptidases Proteins 0.000 description 5
- 239000004365 Protease Substances 0.000 description 5
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 5
- 159000000007 calcium salts Chemical class 0.000 description 5
- 229920001436 collagen Polymers 0.000 description 5
- 210000002196 fr. b Anatomy 0.000 description 5
- 230000001965 increasing effect Effects 0.000 description 5
- 235000021317 phosphate Nutrition 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 4
- 241000283690 Bos taurus Species 0.000 description 4
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 4
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000007796 conventional method Methods 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 235000013861 fat-free Nutrition 0.000 description 4
- 239000000796 flavoring agent Substances 0.000 description 4
- 235000019634 flavors Nutrition 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 4
- 239000010452 phosphate Substances 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 229940104230 thymidine Drugs 0.000 description 4
- 210000000689 upper leg Anatomy 0.000 description 4
- 241000282414 Homo sapiens Species 0.000 description 3
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 3
- QYSXJUFSXHHAJI-XFEUOLMDSA-N Vitamin D3 Natural products C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C/C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-XFEUOLMDSA-N 0.000 description 3
- 238000010171 animal model Methods 0.000 description 3
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 3
- 238000000909 electrodialysis Methods 0.000 description 3
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 3
- 229960002591 hydroxyproline Drugs 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 3
- QYSXJUFSXHHAJI-YRZJJWOYSA-N vitamin D3 Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C\C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-YRZJJWOYSA-N 0.000 description 3
- 235000005282 vitamin D3 Nutrition 0.000 description 3
- 239000011647 vitamin D3 Substances 0.000 description 3
- 229940021056 vitamin d3 Drugs 0.000 description 3
- 208000008035 Back Pain Diseases 0.000 description 2
- 208000010392 Bone Fractures Diseases 0.000 description 2
- 108090000317 Chymotrypsin Proteins 0.000 description 2
- 229920002261 Corn starch Polymers 0.000 description 2
- 102000002322 Egg Proteins Human genes 0.000 description 2
- 108010000912 Egg Proteins Proteins 0.000 description 2
- 206010017076 Fracture Diseases 0.000 description 2
- 208000008930 Low Back Pain Diseases 0.000 description 2
- 108090000284 Pepsin A Proteins 0.000 description 2
- 102000057297 Pepsin A Human genes 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- YZCKVEUIGOORGS-NJFSPNSNSA-N Tritium Chemical compound [3H] YZCKVEUIGOORGS-NJFSPNSNSA-N 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 230000037118 bone strength Effects 0.000 description 2
- 229910000019 calcium carbonate Inorganic materials 0.000 description 2
- MKJXYGKVIBWPFZ-UHFFFAOYSA-L calcium lactate Chemical compound [Ca+2].CC(O)C([O-])=O.CC(O)C([O-])=O MKJXYGKVIBWPFZ-UHFFFAOYSA-L 0.000 description 2
- 239000001527 calcium lactate Substances 0.000 description 2
- 229960002401 calcium lactate Drugs 0.000 description 2
- 235000011086 calcium lactate Nutrition 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 229960001231 choline Drugs 0.000 description 2
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 2
- 229960002376 chymotrypsin Drugs 0.000 description 2
- 239000002285 corn oil Substances 0.000 description 2
- 235000005687 corn oil Nutrition 0.000 description 2
- 239000008120 corn starch Substances 0.000 description 2
- 238000011033 desalting Methods 0.000 description 2
- 210000003278 egg shell Anatomy 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 235000019534 high fructose corn syrup Nutrition 0.000 description 2
- 239000000413 hydrolysate Substances 0.000 description 2
- 229910052500 inorganic mineral Inorganic materials 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 235000010755 mineral Nutrition 0.000 description 2
- 239000011707 mineral Substances 0.000 description 2
- 230000011164 ossification Effects 0.000 description 2
- 210000001672 ovary Anatomy 0.000 description 2
- 229940111202 pepsin Drugs 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 230000035882 stress Effects 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 229910052722 tritium Inorganic materials 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- 229960001322 trypsin Drugs 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- LPZOCVVDSHQFST-UHFFFAOYSA-N 2-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]-3-ethylpyrazol-1-yl]-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C=1C(=NN(C=1)CC(=O)N1CC2=C(CC1)NN=N2)CC LPZOCVVDSHQFST-UHFFFAOYSA-N 0.000 description 1
- BGNGWHSBYQYVRX-UHFFFAOYSA-N 4-(dimethylamino)benzaldehyde Chemical compound CN(C)C1=CC=C(C=O)C=C1 BGNGWHSBYQYVRX-UHFFFAOYSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 208000006386 Bone Resorption Diseases 0.000 description 1
- 102000055006 Calcitonin Human genes 0.000 description 1
- 108060001064 Calcitonin Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000007059 acute toxicity Effects 0.000 description 1
- 231100000403 acute toxicity Toxicity 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 230000024279 bone resorption Effects 0.000 description 1
- 235000008429 bread Nutrition 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- BBBFJLBPOGFECG-VJVYQDLKSA-N calcitonin Chemical compound N([C@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(N)=O)C(C)C)C(=O)[C@@H]1CSSC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1 BBBFJLBPOGFECG-VJVYQDLKSA-N 0.000 description 1
- 229960004015 calcitonin Drugs 0.000 description 1
- 235000010216 calcium carbonate Nutrition 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 230000003913 calcium metabolism Effects 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 235000013351 cheese Nutrition 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- YMFBFFPJRABBPE-BTVCFUMJSA-N ethanol;(2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanal Chemical compound CCO.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O YMFBFFPJRABBPE-BTVCFUMJSA-N 0.000 description 1
- 210000003918 fraction a Anatomy 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 239000008274 jelly Substances 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 235000021243 milk fat Nutrition 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 235000012149 noodles Nutrition 0.000 description 1
- 210000004798 organs belonging to the digestive system Anatomy 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 235000020122 reconstituted milk Nutrition 0.000 description 1
- 229940108461 rennet Drugs 0.000 description 1
- 108010058314 rennet Proteins 0.000 description 1
- 235000013580 sausages Nutrition 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 235000014347 soups Nutrition 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
- C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S426/00—Food or edible material: processes, compositions, and products
- Y10S426/80—Geriatric
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S426/00—Food or edible material: processes, compositions, and products
- Y10S426/807—Poultry or ruminant feed
Abstract
An osteoblast growth promoting and bone reinforcing factor comprising either a precipitate prepared by adjusting the pH of whey or an aqueous solution of whey proteins to 2 to 6 and the concentration of ethanol to 10 % or above, or a water-soluble component prepared by dissolving the precipitate in water and having a molecular weight of 5,000-28,000 Da. The above factor having a molecular weight of 5,000-28,000 Da and an isoelectric point of 4 to 9 and obtained from a supernatant of heated whey or aqueous solution of whey proteins, a precipitate formed by treating the same with sodium chloride or a permeate formed by the ultrafiltration thereof. Food, medicine and feed containing the above factor and useful for preventing and treating osteoarthropathy.
Description
SU(
New Zealand No. 246211 International No. PCT/JP92/01699
TO BE ENTERED AFTER ACCEPTANCE AND PUBLICATION
Priority dates: ->. -s ■ ^ z
International filing date: is • t-z--
Classification: PUfc-b^/xo; e^nuusfou
Publication date: 22 DEC 199^
Journal No.: | ^ $-)
NEW ZEALAND PATENTS ACT 1953
COMPLETE SPECIFICATION
Title of invention: Bone enhancing factors and foods and drinks containing the same
Name, address and nationality of applicant(s) as in international application form:
SNOW BRAND MILK PRODUCTS CO LTD, 1-1, Naebocho 6-chome, Higashi-ku, Sapporo-shi, Hokkaido, JAPAN.
I. (F»llo\*eJ Iffy IA
24 6 2 1
BONE ENHANCING FACTORS AND FOODS AND DRINKS CONTAINING THE SAME
1 . FIELD OF THE INVENTION
This invention relates to an osteoblast growth and bone enhancing factor(s) derived from whey. Further, this invention relates to a bone enhancing factor(s) and foods containing the factor(s). Foods, feeds and medicines containing the factor(s) make bones of human who takes them strong and are useful for preventing or treating maladie in joints of bones disease and osteoporosis.
2 . BACKGROUND OF THE INVENTION
Recently, the number of patients suffering from bone maladie such as osteoporosis, fracture and lumbago is increasing in proportion to increase of aged people. These bone maladie are mainly caused by lack of calcium intake, lowering of calcium absorbing ability, lack of secretion of active vitamin D3, unbalance of hormones relating calcium metabolism and the like and the main causes are made clear as follows.
In bone tissues, bones are always destroyed, resorbed and created. In young age, the bone resorption and formation are balanced, but the bone,7
■j '
"-1SEPI9915
- lA .'
24 6 2
resorbing activity in the bone tissres becomes higher than the bone creating activity in proportion to aging (hereinafter called uncoupling) by various causes. When the uncoupling continues for a long time, the bone tissues become brittle and the above mentioned maladie are caused. Therefore, it is considered that the osteoporosis, fracture and lumbago can be prevented by preventing the uncoupling.
As treatments and prevention against the maladie in joints of bones and osteoporosis by preventing the uncoupling, (1) sufficient calcium supply by foods, (2) light exercises, (3) sun tan and (4) medical treatments have been performed in past.
For the calcium supply by foods, calcium salts such as calcium carbonate, calcium lactate, calcium phosphate and the like and natural calcium materials such as bovine bone powder, egg shell powder, fish bone powder and the like are used. However, some of these calcium salts become water insoluble in digestive organs and cannot be absorbed from intestines and absorbed calcium is not always used for bone formation, if bone creating activities are low.
As the light exercises, a gate ball, light
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walk for arround 30 minutes or the like is considered to be recommendable. However, as a body becomes weak, it becomes hard to do light exercises. Furthermore, it is almost impossible to do exercises for aged people who cannot even get up from a bed.
Sun tan is good for supply of vitamin D3, but the supply of vitamin D3 is not enough to prevent the uncoupling and to prevent the maladie in joints of bones and osteoporosis.
For the medical treatments, medicines such as 1 or-hydroxyvitamin D3, calcitonin and the like are popularly known for bone enhancing ability themselves. However, these medicines consists of chemical compounds as effective components and are not food materials which can enhance bones by being taken safely in a mild condition for a long time.
As mentioned above, the food materials taken safely in a mild condition for a long time to enhance bones, whose effect is proved and established, are not known yet.
3 . DISCLOSURE OF THE INVENTION
Milk is well known as a safe food to be taken for a long time and to enhance bones. The present inventors took notice of this nature of milk,
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assumed that there would be a factor(s) to enhance bones in milk, particularly whey and have been studying about the bone enhancing factor(s) in whey proteins. That is, the inventors have been trying to fractionate a protein fraction in milk, particularly whey and to extract a fraction having osteoblast growth and bone enhancing ability from the protein fraction. Consequently, the inventors found that a mixture of proteins and peptides obtained by treating a water soluble fraction of whey proteins with reverse osmosis (RO), electrodialysis (ED) or the like to remove salts contained in whey proteins had bone enhancing ability and applied the invention as Japanese Patent Application HEI 2-309504.
The inventors have been carrying out the fractionation of such whey proteins, found that the fraction clarified in physical properties by the further fractionation contained the factor(s)
having osteoblast growth and bone enhancing ability and reached to the present invention.
Accordingly, the main object of the present invention is to separate and extract the fraction having bone enhancing ability from milk,
particularly whey and to provide the osteoblast.
growth and bone enhancing factor(s). ^
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The further object of this invention is to provide foods and drinks, medicines and feeds (hereinafter called foods and so on) containing the factor(s) which are capable of enhancing bones and preventing or treating maladie in joints of bones and osteoporosis.
As mentioned above, the inventors further tried to carry out the fractionation of whey proteins and found that the following fraction contained the osteoblast growth and bone enhancing factor(s). That is, this invention relates to the osteoblast growth and bone enhancing factor(s) shown as follows.
This invention provides the osteoblast growth and bone enhancing factor(s) existing in whey,
which is obtained by precipitating whey or whey protein aqueous solution in a final ethanol concentration of more than 10£ within the range of pH from 2 to 6 and has a molecular weight of 5,000 to 28,000 dalton (by SDS-polyacrylamide gel electrophoresis).
This invention also provides the osteoblast growth and bone enhancing factor(s) existing in whey, which is obtained by precipitating whey or whey protein aqueous solution in a final ethanol concentration of more than 10^ within the range of
"" JSEP1993
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pH from 2 to 6 and extracting a water soluble fraction from the precipitate and has a molecular weight of 5,000 to 28,000 dalton (by SDS-polyacrylamide gel electrophoresis).
Further, this invention relates to the following osteoblast growth and bone enhancing factor(s) mentioned below particularly.
This invention provides the osteoblast growth and bone enhancing factor(s) existing in whey,
which is obtained by filtering whey or whey protein aqueous solution by an ultrafilter capable of fractionating substances with a molecular weight of more than 30,000 and extracting a water soluble fraction permeated and has a molecular weight of 5,000 to 28,000 dalton (by SDS-polyacrylamide gel electrophoresis) and an isoelectric point of U to 9.
This invention also provides the osteoblast growth and bone enhancing factor(s) existing in whey, which is obtained by heating whey, whey protein aqueous solution or its water soluble fraction permeated through an ultrafilter capable of fractionating substances with a molecular weight of more than 30,000, at 80 TC for 10 minutes and extracting a supernatant and has a molecular weight of 5,000 to 28,000 dalton (by SDS-polyacrylamide gel
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electrophoresis) and an isoelectric point of 4 to 9.
This invention further provides the osteoblast growth and bone enhancing factor(s) existing in whey, which is obtained by precipitating whey, whey protein aqueous solution or its water soluble fraction permeated through an ultrafilter capable of fractionating substances having a molecular weight of at more than 30,000, in a final NaCl concentration of more than 0.2M within the range of pH from 1.5 to 3.5 to produce a precipitate and extracting a water soluble fraction from the precipitate and has a molecular weight of 5, 000 to 28, 000 dalton (by SDS-polyacrylamide gel electrophoresis) and an isoelectric point of 4 to 9.
This invention still further provides the osteoblast growth and bone enhancing factor(s) existing in whey, which is obtained by heating whey or whey protein aqueous solution at 80*C for 10 minutes so as not to produce a precipitate, filtering a supernatant by an ultrafilter capable of fractionating substances with a molecular weight of more than 30,000 and extracting a permeate and has a molecular weight of 5,000 to 28,000 dalton (by SDS-polyacrylamide gel electrophoresis) and an
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isoelectric point of 4 to 9.
This invention still further provides the osteoblast growth and bone enhancing factor(s)
existing in whey, which is obtained by treating whey or whey protein aqueous solution in a final NaCl concentration of more than 0. 2M within the range of pH from 1.5 to 3.5 to produce a precipitate, filtering a water soluble fraction in the precipitate by an ultrafilter capable of fractionating substances with a molecular weight of more than 30, 000 and extracting a permeate and has a molecular weight of 5, 000 to 28, 000 dalton (by SDS-polyacrylamide gel electrophoresis) and an isoelectric point of 4 to 9.
Furthermore, this invention relates to foods and drinks, medicines and feeds containing the osteoblast growth and bone enhancing factor(s).
The osteoblast growth and bone enhancing factor(s) of the present invention can be obtained by adding ethanol to whey or whey protein aqueous solution to produce a precipitate and extracting a water soluble fraction contained in the precipitate.
It is preferable to carry out the ethanol precipitation in a final ethanol concentration of more than 10% within the range of pH from 2 to 6 to obtain the fraction having a molecular weight of
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, 000 to 28, 000.
»
Further, the osteoblast growth and bone enhancing factor(s).can be obtained by heating whey or whey protein aqueous solution at 80*0 for 10 minutes, removing the formed precipitate and treating the resulting supernatant with an ultrafilter or other filters capable of fractionating substances with a molecular weight of more than 30,000 and extracting the fraction permeated. Also, the factor(s) can be obtained by treating whey or whey protein aqueous solution in a final NaCl concentration of more than 0. 2M under an acidic condition, preferably pH 1.5~3. 5, to produce a precipitate, extracting a water soluble fraction contained in the precipitate, treating the water soluble fraction with an ultrafilter or other filters capable of fractionating substances with a molecular weight of more than 30,000 and extracting the fraction permeated. Further, the factor(s) can be obtained by treating whey or whey protein aqueous solution with an ultrafilter capable of fractionating substances with a molecular weight of more than 30,000 to separate the fraction permeated and treating the fraction under heating or with NaCl. The thus obtained fraction has a molecular weight of 5,000 to 28,000 dalton (by SDS-
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polyacry1 amide gel electrophoresis ) and an isoelectric point of 4 to 9.
As will be detailed in the following examples, the fraction has osteoblast growth and bone enhancing ability and is regarded as the osteoblast growth and bone enhancing factor(s).
Further, this invention relates to foods and drinks, medicines and feeds containing the thus obtained osteoblast growth and bone enhancing factor (s).
A raw material of the osteoblast growth and bone enhancing factor(s) is milk.
Usable as milk in the present invention are fresh milk, milk powder, non-fat milk and reconstituted milk of cows, human beings, goats and sheeps. It is preferable to use whey proteins extracted from whey obtained from said milk.
The whey is transparent yellow-green liquid obtained by adding an acid or rennet to milk or non-fat milk and removing the formed coagulate and is obtained as a by-product of cheese or casein manufacturing. In the other hand, the whey proteins are produced by subjecting said whey to an ultrafiltration, reverse osmosis, chromatography,
dialysis and the like and contains whey protein concentrate having high protein content, obtained
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by removing lactose. The whey proteins to be used in the present invention may be a peptides or a hydrolyzed product of whey proteins by a protease.
In the present invention, the proteins and peptides obtained as a precipitate by fractionating whey proteins with ethanol is used and in the fractionation, the final concentration of ethanol is more than 10% and pH of the solution is 2 to 6, preferably.
For instance, the osteoblast growth and bone enhancing factor(s) of the present invention is produced as follows. First, whey or whey proteins (preferably whey protein concentrate or peptides obtained by hydrolyzing whey proteins with a protease or so on) is dissolved in water, pH of the solution is adjusted from neutral condition to acidic condition of 2 to 6 and ethanol is added to the solution so that the final concentration of ethanol becomes more than 10%, preferably 10~ 60%, to produce a precipitate. The resulting precipitate can be used in the present invention and preferably,
a water soluble fraction obtained by adding water to the precipitate is used to obtain the osteoblast growth and bone enhancing factor(s) having higher activity. Ordinarily, the water soluble fraction is lyophilized into powder. The water soluble fraction
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has the main band within the range of molecular weight from 5,000 to 28,000 by SDS-polyacrylamide gel electrophoresis.
Even when the precipitated fraction is hydrolyzed by a protease such as pepsin, trypsin, chymotrypsin or the like, the resulting hydrolysate product still has the osteoblast growth and bone enhancing ability.
Furthermore, the osteoblast growth and bone enhancing factor(s) of the present invention is contained in the supernatant obtained by heating said whey or whey protein aqueous solution at 80"C for 10 minutes and removing the formed precipitate and is contained in a water soluble fraction of the precipitate obtained by treating said whey or whey protein aqueous solution in a final NaCl concentration of more than 0.2M within the range of pH from 1. 5 to 3. 5.
The present inventors examined the heat stability of whey or whey proteins by heat treatment. As the result, the osteoblast growth and bone enhancing activity was not reduced by the heat treatment at 80TC for 10 minutes. However, the activity was reduced to approximately 85% by the heat treatment at 90 *C for 10 minutes and to approximately 70% by the heat treatment at 951C for
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minutes. The osteoblast growth and bone enhancing factor(s) can be separated from other components in whey by using these properties of the heat stability. In other words, the heat unstable fraction can be removed as the precipitate by carrying out the heat treatment and pH value of the heat treatment, which can separate the heat unstable fraction effectively is 3.5 to 7.0.
NaCl treatment of the present invention can be carried out effectively within the range of pH from 1. 5 to 3. 5. In the fractionation by NaCl, the formation of precipitate is different according to each pH value, but the precipitate is formed in final NaCl concentration of more than 0. 2M within the range of pH from 3.0 to 3.5. Other sodium salts, pottasium salts, ammonium salts, phosphates, bivalent metal salts or the like may be used instead of NaCl and in these cases, the concentration of salts and pH value should be changed according to each condition.
It was found that the osteoblast growth and bone enhancing factor(s) of the present invention passed through an ultrafilter capable of fractionating substances with a molecular weight of more than 30,000. Usually, the resulting water soluble fraction is lyophilized into powder. The
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water soluble fraction has the main band within the range of molecular weight from 5,000 to 28, 000 by SDS-polyacrylamide gel electrophoresis.
Even when the precipitated fraction is subjected to limitative hydrolysis by a protease such as pepsin, trypsin, chymotrypsin or the like, the resulting hydrolysate still has the osteoblast growth and bone enhancing ability.
In the present invention, the osteoblast growth and bone enhancing factor(s) can be contained in foods and so on.
It is preferable to add an absorptive calcium salt(s) such as calcium chloride, calcium carbonate, calcium lactate, natural calcium salt derived from egg shell or milk and others containing the same to the foods and so on containing the osteoblast growth and bone enhancing factor(s).
Further, the whey proteins or ethanol fractionation product thereof may be subjected to desalting treatment.
That is, the foods and so on of the present invention contain the fractionation product of whey proteins, desalted fraction or protease treated fraction thereof and the absorptive calcium salt (as the case may be).
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As the foods and so on containing the osteoblast growth and bone enhancing factor(s) of the present invention, milk, juice, jelly, bread,
noodles, soup, sausage, tablet and the like are exemplified. As the feeds, feed additives or other feeds and as the medicines, a tablet, granular agent, liquid agent and the like administrative orally are exemplified.
Human bones can be enhanced by taking foods t
and so on containing 100 ~500mg of the osteoblast growth and bone enhancing factor(s) of the present invention per one day.
Acute toxicity was not recognized as the result of rat toxicity test, because the fraction was made from milk.
4 . BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the osteoblast growth effect of ethanol fractionation product of whey proteins and peptides in Example 3. When the osteoblast growth activity in case of culturing osteoblast cells in a conventional medium only is assumed 100%, the osteoblast growth activity in case of adding the fractionation product of whey proteins in final ethanol concentration of 10, 20, 40 and 60%.
to the medium is shown.
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Figure 2 shows the bone enhancing ability of ethanol fractionation product of whey proteins and peptides in Example 4. The result of measuring bone breaking stress against rat femurs is shown.
Figure 3 shows the bone enhancing ability of ethanol fractionation product of whey proteins and peptides in Example 4. The result of measuring bone breaking energy against rat femurs is shown.
Figure k shows the osteoblast growth effect in Example 13«
Figure 5 shows the acceleration effect of collagen synthesis in Example 14.
Figure 6 shows the bone breaking force of osteoporosis model rats in Example 15.
Figure 7 shows the bone breaking energy of osteoporosis model rats in Example 16.
. BEST MODE FOR PRACTICE OF THE INVENTION
In the examples herein, the production of whey proteins of the present invention is described.
EXAMPLE 1. PREPARATION OF ETHANOL FRACTIONATION PRODUCT OF WHEY PROTEINS FROM WHEY PROTEIN CONCENTRATE Whey protein concentrate was dissolved in distilled water so as to give a concentration of
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%. To 3 I of the resulting solution, ethanol was added in the amount of 0. 33 & , 0. 75 I , 2 £ and 4. 5 i each so that the final concentration in the fractionation became 10%, 20%, 40% and 60% each.
The mixed solution was stirred enough and left to stand at 41C for 3 days. The resulting solution was centrifuged at 2,500G for 15 minutes to fractionate into supernatant and precipitate. To the precipitate, 500 «£ of distilled water was added and the resulting solution was centrifuged at 2, 500G for 15 minutes to obtain a water soluble fraction. The fraction was lyophilized to obtain powder containing the ethanol fractionation product of whey proteins and peptides and the yield were 42g, 23g,
7g and 6g in ethanol concentration of 10%, 20%, 40% and 60%, respectively.
As the result of fractionating the powder by SDS-polyacrylamide gel electrophoresis, the main band was found within the range of molecular weight from 5, 000 to 28, 000 in all cases.
EXAMPLE 2 : PREPARATION OF ETHANOL FRACTIONATION PRODUCT OF WHEY PROTEINS FROM UNPASTEURIZED WHEY
6N hydrochloric acid was added to 20 & of unpasteurized whey so as to adjust to pH 4. After
U
*.
4 *
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adding 80 i of ethanol to the resulting solution, the solution was stirred enough and left to stand at 4 TC for 3 days. The thus prepared solution was centrifuged at 2,500G for 15 minutes to fractionate into supernatant and precipitate. To the precipitate, 30 St of distilled water was added and the resulting solution was further centrifuged at 2,500G for 15 minutes to obtain a water soluble fraction. The fraction was desalted and concentrated at the same time by reverse osmosis and lyophilized to obtain 42g of powder containing the ethanol fractionation product of whey proteins and peptides.
As the result of fractionating the powder by SDS-polyacrylamide gel elecrophoresis, the main band was found within the range of molecular weight from 5, 000 to 28, 000.
EXAMPLE 3 : OSTEOBLAST GROWTH EFFECT OF ETHANOL FRACTIONATION PRODUCT OF WHEY PROTEINS AND PEPTIDES Osteoblast cells (MC3T3-E1) were cultured with «-MEM (manufactured by Flow Labolatories) containing 0.3% of bovine blood serum using 96 well culture plate. 10// jg of 5% aqueous solution of the powder obtained in Example 1 was added and the incubation was carried out for 18 hours. Thymidine
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labeled with tritium was added and the osteoblast growth activity was measured by radioactivity of thymidine absorbed into the osteoblast cells after 2 hours.
Figure 1 shows the result of examination. The figure shows that the osteoblast growth activity measured by the radioactivity is increased when the ethanol fractionation product of whey proteins or peptides is added, as compared with no-addition of the ethanol fractionation product. In this case, the radioactivity in no-addition of the ethanol fractionation product is 100%. When the osteoblast cells were incubated with the ethanol fractionation product of whey proteins and peptides obtained in the final ethanol concentration of 10%~60%, the osteoblast growth activity was incresed up to 200~ 300% as compared with the culture using the medium only. From the result, it is clear the ethanol fractionation product of whey proteins have osteoblast growth ability.
EXAMPLE 4 : BONE ENHANCING ABILITY OF ETHANOL FRACTIONATION PRODUCT OF WHEY PROTEINS AND PEPTIDES Table 1 shows a compositon of foods given to experimental animals.
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TABLE 1. Composition of foods used for experiment (g/IOOg)
Sham & Control Whey protein group group
Sucrose
49. 3
49.
3
Casein
18
Corn starch
Cellulose
Corn oil
Vitamins
1. 2
1.
2
(containing choline)
Minerals
4. 5
4.
Ethanol fractionation
-
2
product of whey proteins
As shown in Table 1, 2g of the ethanol fractionation product of whey proteins obtained in Example 2 was added to foods to replace 20g of casein with 18g. Calcium and phosphate were added 300mg/100g to all foods. The ratio of calcium and phosphate was controlled to be 1:1.
SD female rats (7 weeks after birth) were • -
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used as experimental animals. Osteoporosis model rats (OVX rats) were oophorectomized and fed by low calcium foods for a month. At the same time, 7 Sham rats were operated in the same way as above without taking out ovaries (pretended operation). Experiments were carried out by 7 rats as one group each.
One group of the osteoporosis model rats was fed by control foods and another group was fed by foods containing the ethanol fractionation product of whey proteins, shown in Table 1, supra, for a month.
After a month, femurs of all rats in each group were taken out and the bone strength of them was measured using a breaking force measuring equipment. The result was shown in figure 2 and 3. As shown in figure 2, the bone breaking stress of the rat group fed by whey protein foods was significantly higher than the group fed by control foods. The bone breaking energy was also high value as shown in figure 3« From the result, it is clear that the ethanol fractionation product of whey proteins have bone enhancing ability. Next, foods containing the ethanol fractionation product of whey proteins obtained in Example 2 are datailed in the following examples. • • .
t
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EXAMPLE 5 : DRINKS CONTAINING ETHANOL
FRACTIONATION PRODUCT OF WHEY PROTEINS
(weight f>)
High fructose corn syrup 15.0
Juice 10. 0
Citric acid 0. 5
Ethanol fractionation 0. 5 Product of whey proteins
Flavor 0. 1
Calcium 0. 1
Water 73.8
The above components were mixed, filled in a container and pasteurized by heating to obtain drinks by a conventional method.
EXAMPLE 6 : TABLETS CONTAINING ETHANOL
FRACTIONATION PRODUCT OF WHEY PROTEINS
(weight %)
Water containing crystalline glucose Ethanol fractionation product of whey proteins
73.5 20. 0
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Calcium 5.0
Sugar ester 1. 0
Flavor 0.5
The above components were mixed and pressed to make tablets by a conventional method.
EXAMPLE 7
15kg of whey protein concentrate was dissolved in water to prepare 100 jg of the whey protein solution. The resulting solution was adjusted to pH 4.5 and heated by steam at 80'C for 10minutes. The solution was centrifuged at 5, 000G for 10 minutes to remove the precipitate and to obtain 80 £ of the solution. 10 £ of the solution was lyophilized to obtain 530g of powder (Fraction A). Separately, 35 £ of the remaining solution was adjusted to pH 3.0 and after adding NaCl so as to give a concentration of 0.2M, centrifuged at 15, 000G for 10 minutes to remove the precipitate. A NaCl concentration of the resulting supernatant was adjusted to 2.0M and the formed precipitate was collected by a centrifugal separation at 15,000G for 10 minutes. The precipitate was suspended in 20 £ of distilled water and 10 £ of the resulting
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solution was desalted by reverse osmosis (RO) and lyophilized to obtain powder (Fraction B). 10 I of the remaining solution was filtered by an ultrafilter (manufactured by DDS Ltd. ) capable of fractionating substances with a molecular weight of 100,000 and the permeate was filtered again by an ultrafilter capable of fractionating substances with a molecular weight of 30,000. The resulting permeate was desalted by electrodialysis and lyophilized to obtain powder (Fraction C). The yield of Fraction B and of Fraction C were 1, 227g and 540g, respectively.
EXAMPLE 8
7.5kg of whey protein concentrate was dissolved in water so as to give a total amount of 50 & and the solution was adjusted to pH 4. 5 and heated by steam at 80TC for 10 minutes. The resulting solution was filtered by an ultrafilter (manufactured by DDS Ltd. ) capable of fractionating substances with a molecular weight of 500,000 and a half amount (30 £ ) of the permeate was desalted by reverse osmosis (RO) and lyophilized to obtain powder (Fraction D). 30 I of the remaining permeate was adjusted to pH 3.5 and after adding NaCl so as to give a final concentration of 0.3M, centrifuged
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at 1 5, 000G for 10 minutes to remove the formed precipitate. The resulting supernatant was adjusted to NaCl concentration of 2. 0M and centrifuged at 15, OOOG for 10 minutes. The resulting precipitate was separated and lyophilized to obtain powder (Fraction E). The yield of Fraction D and of Fraction E were 632g and 235g, respectively.
EXAMPLE 9
Hydrochloric acid was added to 200 I of nonfat milk till pH of the non-fat milk became 4.7 to precipitate casein and the solution was centrifuged at 5, OOOG for 20 minutes to obtain 185 t of whey. The whey was filtered by an ultrafilter (manufactured by DDS Ltd.) capable of fractionating substances with a molecular weight of 500,000 and the resulting permeate was further filtered by an ultrafilter capable of fractionating substances with a molecular weight of 30,000 to carry out the desalting and concentration and to obtain 6 jI of concentrate. 2 £ of the concentrate was lyophilized as it was to obtain powder (Fraction F). 2 £ of the concentrate was heated at 80 "C for 10 minutes under pH 6.3 and centrifuged at 5, OOOG for 10 minutes. The resulting supernatant was lyophilized to obtain powder (Fraction G). 2 t of the remaining. _•
/
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concentrate was adjusted to pH 1.5 and a NaCl concentration of 3M and centrifuged at 15,OOOG for 10 minutes. The resulting precipitate was dissolved in water and the solution was desalted by electrodialysis and lyophilized to obtain powder (Fraction H). The yield of Fraction F, of Fraction H and of Fraction G were 340g, 188g and 20 2g, respectively.
EXAMPLE 10
. 0kg of whey protein concentrate was dissolved in 50 £ of water and the solution was adjusted to pH 3. 5 and a final NaCl concentration of 3. 0M. The formed precipitate was recovered by a centrifugal separation at 15, OOOG for 10 minutes and dissolved in 10 £ of water. 2 £ of the resulting solution was electrodialyzed.and lyophilized to obtain powder (Fraction I). 8 £ of the remaining solution was adjusted to pH 4. 5 and heated at 80"C for 10 minutes. A half amount of the heat-treated solution was centrifuged at 5,OOOG for 10 minutes and the resulting supernatant was lyophilized to obtain powder (Fraction J). The remaining heat-treated solution was filtered by an ultrafilter capable of fractionating substances with a molecular weight of 500, 000 and the permeate was - •.
• c
/ ■< ■
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desalted by reverse osmosis (RO) and lyophilized to obtain powder (Fraction K). The yield of Fraction I, of Fraction J and of Fraction K were 430g, 255g and 178g, respectively.
EXAMPLE 11
2. 5kg of whey protein concentrate was dissolved in 50 £ of water and the solution was adjusted to pH 3.0 and a final NaCl concentration of 3. 0M. The formed precipitate was recovered by a centrifugal separation at 15,OOOG for 10 minutes and dissolved in 10 I of water. The solution was filtered by an ultrafilter capable of fractionating substances with a molecular weight of 500,000. A half amount of the resulting permeate was desalted by reverse osmosis (RO) and lyophilized to obtain powder (Fraction L). The remaining permeate was heated at 80 "C for 10 minutes and centrifuged at 5, OOOG for 10 minutes and the resulting supernatant was lyophilized to obtain powder (Fraction M). The yield of Fraction L and of Fraction M were 216g and 175g, respectively.
EXAMPLE 12
Lyophilized Fractions A~M obtained in Examples 7~ 11 were fractionated by SDS-
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polyacrylamide gel electrophoresis. The main band was found within the range of molecular weight from 5,000 to 28,000 in all cases. The same Fractions were subjected to isoelectric electrophoresis and the main band was found within the range of isoelectric point from 4 to 9 in all cases.
EXAMPLE 13 : OSTEOBLAST GROWTH EFFECT OF FRACTIONS A~M OBTAINED IN EXAMPLES 7~11 Osteoblast cells (MC3T3-E1) were cultured with or-MEM (manufactured by Flow Labolatories) containing 0. 3% of bovine blood serum using 96 well culture plate.
Fractions A~M obtained in Examples 7 — 11, in particular were particularly added to the wells so that the final concentration became 5 (i g/ m SL and 50 jj g/ m £ and the incubation was carried out for 18 hours. Thymidine labeled with tritium was added and each osteoblast growth activity was measured by radioactivity of thymidine absorbed into the osteoblast cells after 2 hours.
Figure 4 shows the result of examinations. The figure shows that the osteoblast growth activity measured by the radioactivity is increased when Fractions A ~ M, in particular are added, as compared with no-addition of Fractions A~ M. In i » /
a
-v
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this case, the radioactivity in no-addition of Fractions A ~ M is 100%.
When the osteoblast cells were incubated with Fractions A ~ M having the final concentration of 50 //g/a£, the osteoblast growth activity was increased up to 150~420% as compared with the culture using the medium only. Even when the final concentration was 5 ug/a£j the osteoblast growth activity was also observed. From the result, it is clear that Fractions A~M obtained in Examples 7 — 11 have osteoblast growth activity.
The same result was obtained when osteoblast cells (UMR 106) were cultured with Fractions A~ M in the same way as above.
EXAMPLE 14 : EFFECT OF STIMULATING COLLAGEN
SYNTHESIS
Osteoblast cells (MC3T3-E1) were cultured with the same medium as Example 13. Fractions A ~M obtained in Examples 7 — 11, in particular were particularly added so as to give a final concentration of 50 // g/»e and the incubation was carried out at 37 "C for 3 days. An amount of synthesized collagen was measured. The amount of collagen was measured by determinating hydroxyproline. The determination of hydroxyproline
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was carried out by adding p-dimethylamino-benzaldehyde to the collapsed cell hydrolyzed solution suspended using 6N hydrochloric acid.
Figure 5 shows the result of examinations. The figure shows that the amount of hydroxyproline is increased when Fractions A ~ M obtained in Examples 7 ~ 11, in particular are particularly added, as compared with the culture using the medium only. From the result, it is clear the collagen synthesis in osteoblast cells is stimulated by the addition of Fractions A ~M.
EXAMPLE 15 : BONE ENHANCING ACTIVITY
Fractions C, E, K and M, in particular were particularly replaced with casein as shown in Table 2, infra, at the amount of 2g to prepare foods. Calcium and phosphate were added to all foods at 300mg/100g respectively foods each and the ratio of calcium and phosphate was controlled to be 1:1.
SD female rats (7 weeks after birth) were used as experimental animals. Osteoporosis model rats were oophorectomized and fed by low calcium foods for a month. At the same time, 7 Sham rats were operated in the same way as above without taking out ovaries (pretended operation). Experiments were carried out by 7 rats as one group
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Experiments were carried out by 7 rats as one group each.
Osteoporosis model rats were fed by each feeds as shown in Table 2, infra.
After a month, femurs of all rats in each group were taken out and the bone strength of them was measured using a breaking force measuring equipment.
TABLE 2 Composition of feeds used in examinations (g/100g)
Fraction
Control Sham C E K M
Sucrose
49. 3
49.3
49.
3
Casein
. 0
.0
18.
0
Corn starch
. 0
.0
.
0
Cellulose
. 0
.0
.
0
Corn oil
. 0
.0
.
0
Vitamins
1. 2
1. 2
1.
2
(containing choline)
Minerals
4. 5
4.5
4.
Fraction C
2.
0
E K
49. 3
49.
3
49.
3
18. 0
18.
0
18.
0
. 0
.
0
.
0
. 0
.
0
.
0
. 0
.
0
.
0
1. 2
1.
2
1.
2
4. 5
4.
4.
2. o
2.0
'-I SEPI993 ■ %
24 6 2 1
M 2. 0
The result of the examinations is shown in Figure 6 and 7. As shown in Figure 6, the bone breaking force of the rats fed by foods containing Fraction C, E, K or M were significantly higher than the rats fed by control foods. The bone breaking energy was also high value in Fraction C, E, K and M groups as shown in Figure 7. From the result, it is clear that the Fraction C, E, K and M have bone enhancing ability.
Next, foods containing Fraction B obtained in Example 7 are detailed in the following examples.
EXAMPLE 16 : DRINKS
Component weight %
High fructose corn syrup 15.0
Juice 10.0
Citric acid 0.5
Fraction B 0. 5
Flavor 0. 1
Calcium 0. 1
Water 73.8
- JSEPI993
24 6 2 1
The above components were mixed, filled in a container and pasteurized by heating to manufacture drinks by a conventional method.
EXAMPLE 17 : TABLETS
Component weight %
Water containing 73. 5 crystalline glucose
Fraction B 20. 0
Calcium 5.0
Sugar ester 1. 0
Flavor 0. 5
The above components were mixed and pressed to make tablets by a conventional method.
6 . INDUSTRIAL APPLICABILITY
Foods and the like containing the osteoblast growth and bone enhancing factor(s) of the present invention have bone enhancing ability and are useful for preventing or treating various maladie in joints of bones and osteoporosis.
'■-istpim
Claims (13)
1 . An osteoblast growth and bone enhancing factor existing in whey, which is obtained by precipitating whey or whey protein aqueous solution in a final ethanol concentration of more than 10% within the range of pH from 2 to 6 and has a molecular weight of 5, 000 to 28, 000 dalton (by SDS-polyacrylamide gel electrophoresis).
2 . An osteoblast growth and bone enhancing factor existing in whey, which is obtained by precipitating whey or whey protein aqueous solution in a final ethanol concentration of more than 10% within the range of pH from 2 to 6 and extracting a water soluble fraction from the precipitate and has a molecular weight of 5,000 to 28,000 dalton (by SDS-polyacrylamide gel electrophoresis).
3 . An osteoblast growth and bone enhancing factor existing in'whey, which is obtained by filtering whey or whey protein aqueous solution by an ultrafilter capable of fractionating substances with a molecular weight of more than 30,000 and extracting a water soluble fraction permeated and has a molecular weight of 5,000 to 28,000 dalton (by SDS-polyacrylamide gel electrophoresis) and an': 1 SEP1993 •: 24 6 2 isoelectric point of 4 to 9.
4 . An osteoblast growth and bone enhancing factor existing in whey, which is obtained by heating whey, whey protein aqueous solution or its water soluble fraction permeated through an ultrafilter capable of fractionating substances with a molecular weight of more than 30, 000, at 80 "C for 10 minutes and extracting a supernatant and has a molecular weight of 5,000 to 28, 000 dalton (by SDS-polyacrylamide gel electrophoresis) and an isoelectric point of 1 to 9.
5 . An osteoblast growth and bone enhancing factor existing in whey, which is obtained by precipitating whey, whey protein aqueous solution or its water soluble fraction permeated through an ultrafilter capable of fractionating substances having a molecular weight of more than 30, 000, in a final NaCl concentration of more than 0. 2M within the range of pH from 1.5 to 3-5 to produce a precipitate and extracting a water soluble fraction from the precipitate and has a molecular weight of 5, 000 to 28, 000 dalton (by SDS-polyacrylamide gel electrophoresis) and an isoelectric point of fl.to 9. / ~-l SEPI99J - 3 5 - 24 6 2 1
6 . An osteoblast growth and bone enhancing factor existing in whey, which is obtained by heating whey or whey protein aqueous solution at 80 "C for 10 minutes so as not to produce a precipitate, filtering a supernatant by an ultrafilter capable of fractionating substances with a molecular weight of more than 30, 000 and extracting a permeate and has a molecular weight of 5,000 to 28,000 dalton (by SDS-polyacrylamide gel electrophoresis) and an isoelectric point of 4 to 9.
7 . An osteoblast growth and bone enhancing factor existing in whey, which is obtained by treating whey or whey protein aqueous solution in a final NaCl concentration of more than 0. 2M within the range of pH from 1.5 to 3.5 to produce a precipitate, filtering a water soluble fraction in the precipitate by an ultrafilter capable of fractionating substances with a molecular weight of more than 30, 000 and extracting a permeate and has a molecular weight of 5,000 to 28,000 dalton (by SDS-polyacrylamide gel electrophoresis) and an isoelectric point of U to 9.
8 . Bone enhancing foods and drinks containing the - ! S:?i993„ - 3 6 - "r * - 1 «u3 J •- f osteoblast growth and bone enhancing factor claimed in any of the claims 1 to 7.
9 . An agent for preventing or treating a bone malady which comprises containing the osteoblast growth and bone enhancing factor claimed in any of the claims 1 to 7.
10. Bone enhancing feeds containing the osteoblast growth and bone enhancing factor claimed in any of the claims 1 to 7.
11. An osteoblast growth and bone enhancing factor existing in whey, which is obtained by a process substantially as herein described with reference to the Examples.
12. An agent for preventing or treating a bone malady comprising the osteoblast growth and bone enhancing factor of claim 11.
13. Bone enhancing feeds substantially as herein described with reference to the Examples.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP03356754A JP3092874B2 (en) | 1991-12-26 | 1991-12-26 | Whey-derived osteoblast-proliferating and bone-enhancing fraction and bone-enhanced food, feed, and pharmaceuticals containing the fraction |
| JP14217092A JP3604159B2 (en) | 1992-05-07 | 1992-05-07 | Whey-derived osteoblast proliferation-promoting and bone-strengthening factor and bone-strengthened food, medicine and feed containing the factor |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| NZ246211A true NZ246211A (en) | 1994-12-22 |
Family
ID=26474258
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| NZ246211A NZ246211A (en) | 1991-12-26 | 1992-12-25 | Osteoblast growth and bone enhancing factor(s) derived from whey and foods containing it |
Country Status (9)
| Country | Link |
|---|---|
| US (1) | US5654019A (en) |
| EP (1) | EP0573668B1 (en) |
| AT (1) | ATE163858T1 (en) |
| DE (1) | DE69224741T2 (en) |
| DK (1) | DK0573668T3 (en) |
| ES (1) | ES2115047T3 (en) |
| GR (1) | GR3026572T3 (en) |
| NZ (1) | NZ246211A (en) |
| WO (1) | WO1993012807A1 (en) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3112637B2 (en) * | 1994-09-30 | 2000-11-27 | 雪印乳業株式会社 | Bone strengthener |
| JP2974604B2 (en) * | 1996-01-23 | 1999-11-10 | 雪印乳業株式会社 | Basic protein composition, basic peptide composition and use thereof |
| JP3073439B2 (en) * | 1996-02-08 | 2000-08-07 | 雪印乳業株式会社 | Bone formation promotion and bone resorption inhibitor |
| JP3018313B2 (en) * | 1996-02-23 | 2000-03-13 | 雪印乳業株式会社 | Bone formation promotion and bone resorption inhibitor |
| WO2000019840A1 (en) * | 1998-10-08 | 2000-04-13 | Bioflash | Process for the isolation of milk proteins |
| NZ507335A (en) * | 2000-10-05 | 2004-10-29 | New Zealand Dairy Board | Bone health compositions derived from milk comprising an acidic protein fraction but that does not contain CGMP |
| DE10331202A1 (en) | 2003-07-10 | 2005-03-31 | S.K. Enterprise Gmbh | Use of whey permeate for the treatment of metabolic syndrome |
| DE102006036285A1 (en) * | 2006-08-03 | 2008-02-07 | "S.U.K." Beteiligungs Gmbh | Whey permeate fractions and their use for the prevention and treatment of type 2 diabetes and metabolic syndrome |
| CN101842026B (en) | 2007-11-01 | 2014-02-19 | 雪印惠乳业株式会社 | Bone-strengthening food material |
| KR101441728B1 (en) * | 2007-11-01 | 2014-09-17 | 유키지루시 메그밀크 가부시키가이샤 | Food material for inhibiting bone resorption |
| FR2988566B1 (en) * | 2012-03-28 | 2014-08-08 | Yoplait France | FOOD COMPOSITIONS FOR STIMULATING BONE TISSUE FORMATION |
| ITRM20120368A1 (en) * | 2012-07-30 | 2014-01-31 | Ambiotec Sas | COMPOSITION ABLE TO ACT ON THE MYOD MUSCLE REGULATOR AND OSTEOBLASTI PROLIFERATION |
Family Cites Families (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4143174A (en) * | 1975-07-24 | 1979-03-06 | Beatrice Foods Co. | Food composition containing whey colloidal precipitate |
| US4089987A (en) * | 1976-09-23 | 1978-05-16 | Stauffer Chemical Company | Whipping composition of modified whey protein and additives |
| GB8723094D0 (en) * | 1987-10-01 | 1987-11-04 | Ciba Geigy Ag | Polypeptide growth factor from milk |
| JPH01126062A (en) * | 1987-11-11 | 1989-05-18 | Ricoh Co Ltd | Color correction device for full color reader |
| DE3743440A1 (en) * | 1987-12-21 | 1989-06-29 | Gauri Kailash Kumar | METHOD FOR SEPARATING THE SOLVED AND UNSOLVED INGREDIENTS OF MILK |
| EP0349048A3 (en) * | 1988-06-27 | 1990-07-11 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Osteogenic growth factors derived from regenerating bone marrow |
| JPH0211599A (en) * | 1988-06-30 | 1990-01-16 | Kaken Pharmaceut Co Ltd | Osteoblast proliferation factor |
| JP2605835B2 (en) * | 1988-10-18 | 1997-04-30 | 味の素株式会社 | Osteoporosis treatment |
| JPH02111797A (en) * | 1988-10-20 | 1990-04-24 | Kaken Pharmaceut Co Ltd | Osteoblast proliferation factor |
| JPH02218697A (en) * | 1989-02-21 | 1990-08-31 | Suntory Ltd | Protein having ossific activity |
| DE69029415T2 (en) * | 1989-02-23 | 1997-04-03 | Yissum Res Dev Co | Osteogenic growth factors identified from regenerating bone marrow |
| JP2802436B2 (en) * | 1989-05-19 | 1998-09-24 | 雪印乳業株式会社 | Bone disease treatment / prevention agent |
| JPH03151877A (en) * | 1989-06-02 | 1991-06-28 | Chiron Corp | Bone calcium precipitation factor |
| JP3160862B2 (en) * | 1990-11-15 | 2001-04-25 | 雪印乳業株式会社 | Bone-fortified foods, feeds and pharmaceuticals |
-
1992
- 1992-12-25 DK DK93900432.1T patent/DK0573668T3/en active
- 1992-12-25 NZ NZ246211A patent/NZ246211A/en not_active IP Right Cessation
- 1992-12-25 WO PCT/JP1992/001699 patent/WO1993012807A1/en active IP Right Grant
- 1992-12-25 EP EP93900432A patent/EP0573668B1/en not_active Expired - Lifetime
- 1992-12-25 US US08/107,771 patent/US5654019A/en not_active Expired - Lifetime
- 1992-12-25 ES ES93900432T patent/ES2115047T3/en not_active Expired - Lifetime
- 1992-12-25 AT AT93900432T patent/ATE163858T1/en active
- 1992-12-25 DE DE69224741T patent/DE69224741T2/en not_active Expired - Lifetime
-
1998
- 1998-04-07 GR GR980400764T patent/GR3026572T3/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| DE69224741T2 (en) | 1998-07-02 |
| EP0573668B1 (en) | 1998-03-11 |
| ATE163858T1 (en) | 1998-03-15 |
| EP0573668A4 (en) | 1995-11-02 |
| DK0573668T3 (en) | 1998-04-27 |
| EP0573668A1 (en) | 1993-12-15 |
| ES2115047T3 (en) | 1998-06-16 |
| DE69224741D1 (en) | 1998-04-16 |
| US5654019A (en) | 1997-08-05 |
| WO1993012807A1 (en) | 1993-07-08 |
| GR3026572T3 (en) | 1998-07-31 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| RENW | Renewal (renewal fees accepted) | ||
| RENW | Renewal (renewal fees accepted) | ||
| ASS | Change of ownership |
Owner name: MEGMILK SNOW BRAND CO., JP Free format text: OLD OWNER(S): SNOW BRAND MILK PRODUCTS CO., LTD |
|
| EXPY | Patent expired |